The apical surface of mammalian bladder urothelium is highly specialized as it is almost completely covered by 2D crystals of hexagonally packed 16-nm uroplakin particles forming urothelial plaques. These plaques play important biological functions, in that they contribute to the formation of a strikingly effective permeability barrier. In addition, these plaques may strengthen the physical stability of the urothelial surface thus preventing it from rupturing during bladder distention, and one of the uroplakins, UPIa, may serve as the receptor for the uropathogenic E. coli that causes >85% of the urinary tract infections. Despite recent progress in the biochemical characterization of the uroplakin proteins, many important questions remain unanswered. For example, what are the mechanisms that control whether a uroplakin or a uroplakin heterodimer can exit from the ER? How are the protein machineries including MAL and Rab27b involved in targeting the uroplakins to the apical surface where they function? To answer these questions, we will pursue three specific Aims. In Aim One, we will identify proteins that are associated with normal UPIb that can exit ER without a uroplakin partner vs. UPIb containing transmembrane domain mutations that is retained by ER, and we will study whether UPIIIa, which can bind to UPIb, can rescue mutated UPIb from being trapped in the ER. In Aim Two, we will employ two genetically engineered mouse mutants that are defective in MAL or Rab27b to see whether they are necessary for the exocytosis of the uroplakin-delivering fusiform vesicles, and whether Rab27b functions upstream of MAL. In Aim Three, we will study the functions of the conserved head domain vs. the variable tail domain of the Rab27b and its isoform Rab27a, by adenovirally delivering Rab27a, Rab27b and their domainexchanging chimeras into Rab27b-null umbrella cells. Together, these studies will yield new insights into the mechanism of urothelial membrane trafficking and pave the way for future studies on the possible role(s) of urothelial vesicular transport in bacterial invasion into and release from the urothelial cells.